The polysialic acid of Escherichia coli K1 commonly occurs in invasive E. coli disease. Enzymes involved in its biosynthesis are central to neuraminic acid metabolism in bacteria and mammals. The expression and function of 2 of these enzymes is being investigated, namely, CMP-neuAc synthetase (neuA gene product) and a protein of undefined function, (P7 neuC gene product). CMP-NeuAc synthetase contains two cysteine residues. Site directed mutagenesis of cys-129 or cys-329 to glycine results in loss of activity, suggesting that both residues are important. However, enzyme retains activity when cys-129 is changed to a serine and is therefore not essential for catalysis. Random mutagenesis reveals that cys-329 is essential. The cys-129 to serine (ser-129) results in a less stable enzyme. Both the wild type and the ser-129 enzyme are inactivatable only after partial thermal denaturation with charged sulfhydryl reagents, suggesting a buried residue. Hydrophobic reagents can inactivate both enzymes at 25 degrees C but not in the presence of CTP, suggesting that cys-329 is buried at the active site. To locate common functional domains, monoclonal antibodies were prepared to recombinant CMP-neuAc synthetase. Purification from other sources is underway. Recombinant CMP-neuAc synthetase is being used as a reagent to synthesize sialylated oligosaccharides. The amino terminus of the P7 protein was determined from fusion proteins confirming the suggestion in the previous report that the neuA and neuC genes overlap. Purification of P7 and investigation of its function is underway. Other genes in the K1 cluster: Polysaccharide was isolated from E. coli mutants which do not export polymer. Examination of these polymers by size exclusion, NMR, and TLC suggest kpsT mutants produce polymers similar to wild type but without phosphotidic acid. kpsD mutants produce a polysaccharide with different hydrodynamic behavior.